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Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
EISSN: 1677-3225
Vol. 10, Num. 2, 2011, pp. 120-123

Brazilian Journal of Oral Sciences, Vol. 10, No. 2, Apr-Jun, 2011, pp. 120-123

Healing properties of papain-basedgel on oral ulcers

Manoela Domingues Martins1, Kristianne Porta Santos Fernandes2, Vanessa Cristina Pavesi2, Cristiane Miranda França2, Raquel Agnelli Mesquita-Ferrari2, Sandra Kalil Bussadori2

1Oral Pathology Department, School of Dentistry, Federal University of Rio Grande do Sul, Brazil
2
Graduate Program in Rehabilitation Sciences, Nove de Julho University (UNINOVE), Brazil

Correspondence to: Cristiane Miranda Franca Av. Conselheiro Rodrigues Alves, 948 apto 93 Vila Mariana, São Paulo, SP, Brazil CEP: 04014-002 E-mail: cristiane321@gmail.com

Received for publication: November 23, 2010 Accepted: May 18, 2011

Code Number: os11024

Abstract

The oral mucous membrane is prone to developing ulcers originating from traumatic or immunological processes.
Aim:
The aim of the present study was to perform a histological evaluation of the antiinflammatory and healing properties of PapacarieTM applied to oral ulcers.
Methods
: Fifty adult female albino Wistar rats were randomly separated into two groups: control and PapacarieTM. The animals were anesthetized, placed in prone position and ulcers were induced in the middle dorsum of the tongue through a 3-mm-diameter punch. The control group received no treatment thereafter, while those in the PapacarieTM group received an application of the gel twice a day for 14 days. Five animals from each group were euthanized on days 1, 3, 5, 7 and 14. The tongues were removed, fixed, routinely processed for hematoxylin-eosin staining and analyzed by two blind, calibrated pathologists for the presence or absence of ulcer, inflammatory infiltrate and neutrophils. Data were submitted to statistical analysis by the Fisher's exact test.
Results:
On day 1, both groups exhibited ulceration and dense acute inflammatory infiltrate. On day 5, reepithelization and scanty chronic inflammatory infiltrate were observed. On day 14, all animals were healed. There were no statistically significant differences between groups with regard to ulceration (p=0.81), inflammation (p=0.55) or neutrophils (p=0.53).
Conclusions:
Oral ulcers treated with a papainbased gel exhibited the same inflammatory reaction and healing aspects as those of the nontreated control group.

Keywords: papain, oral ulcer, Wistar, wound healing.

Introduction

The oral mucous membrane is prone to developing ulcers originating from traumatic or immunological processes. These episodes of oral trauma or aphthousattacks are characterized by painful lesions that range from the size of a pinheadup to several centimeters in diameter. There have been many attempts over the years to find an effective treatment for oral ulceration and localized topical regimens are considered to be the standard treatment for mild cases of oral ulcers. Standard topical treatment options that provide relief from symptoms include analgesics,anesthetics, antiseptics, anti-inflammatory agents, steroids, sucralfate, tetracycline suspension and silver nitrate. Dietary modifications may also support therapeutic measures1.

Despite its size, an oral ulcer can cause mild to severe pain. In a recent study on oral health-related quality of life (OHRQoL) measures for oral diseases, Hapa et al. (2010) found that patients with recurrent aphthousstomatitis reported a lower OHRQoL than the general population2.

Papain is a purified protein extracted from the latex ofthe papaya tree (Carica papaya) and is an archetypalnon specific proteinase widely used by Brazilian nurses intraditional medicine. It can be used as phytotherapic agentto remove damaged or necrotic tissue in the treatment of pressure ulcers, gangrene and eschars and as a debriding chemical agent. Papain digests necrotic tissue by breaking down fibrins in the presence of sulfhydryl groups (e.g.,cysteine) without digesting collagen. Papain liquefies theeschar, thereby facilitating the migration of viable cells from the wound edge to the wound cavity. It is also useful in reducing the bacterial load, decreasing exudates and increasing the formation of granulation tissue3-4.

To improve its healing properties, papain has been associated with other substances. Certain formulations contain urea, which is a chaotropic agent that facilitates the action of papain by solubilizing proteins. Others contain a copper chlorophyllin complex, which has been clinically shown to promote the formation of healthy granulation tissue, control inflammation and diminish odor5.

PapacarieTM is a gel containing papain, chloramine and toluidine blue that has been used to chemically soften cariousdentin tissue6-9. Besides the papain bacteriostatic, bactericide and anti-inflammatory properties, the gel presents chloramine,a compound that contains chlorine and ammonia and hasbactericide and disinfectant properties. The purpose of this study was to perform a histological evaluation of the anti inflammatory and healing properties of this papain-based gel applied to oral ulcers.

Materials and methods

Animals

The sample consisted of 50 adult female albino Wistarrats (Rattus norvegicus albinus), with body weight ranging from 250 to 300 g. The animals were obtained from the central animal lodging facility of Nove de Julho University(UNINOVE), Brazil and were maintained at controlled light and temperature conditions with standard chow and water available ad libitum. All experimental procedures were carried out in compliance with the guidelines of the Brazilian College for Animal Experimentation. This study received approval from the UNINOVE Ethics Committee (processnumber 19/2007/CEA/UNIDERP).

Ulcer induction

After weighing, each animal received a single intramuscular injection of 80mg/kg of xylazine (Anasedan,Vetbrands, Jacareí, SP, Brazil) and 10mg/kg of ketamine (Dopalen, Vetbrands). The animal was then placed in proneposition. A 3-mm-diameter punch was made to induce injuryby removing a circular area from the dorsum of the tonguein such a way that the injury was located on the middle portion of the median sagittal plane. To control de depth of the induced ulcers, a rubber stop was placed on the punch inorder to produce ulcers with 5 mm of depth.

Experimental Groups

The animals were randomly separated into two groups.The control group received no treatment. These animals were handled in the same manner as the other group, but withoutthe application of gel and the ulcers were left to heal without interference. The animals in the papain group received the application of PapacarieTM (Fórmula & Ação, São Paulo, SP,Brazil) twice a day with a cotton swab for fourteen days. The lesions were accompanied daily.

Histological analysis

For the histological analysis, 5 animals from each group were euthanized with an overdose of anesthesia on days 1,3, 5, 7 and 14. The tongue was removed, fixed with 10% paraformaldehyde, embedded in paraffin and routinely processed for hematoxylin-eosin staining. The specimens were analyzed by two blind, calibrated pathologists who evaluated the presence or absence of ulcer, inflammatory infiltrate and neutrophils.

Statistical analysis

Data were subjected to statistical analysis by the Fisher's exact test. The nature of the variables studied and the variability of the means was considered using the Bioestat 5.0 software program. The significance level was set at 5%.

Results

Data from the histological analysis are presented in Table 1. On day 1, both groups exhibited ulceration, dense acute inflammatory infiltrate and neutrophils. On day 3, one animal of each group had no ulcer, only acute inflammation, while the others exhibited ulceration and acute inflammation (Figures 1 A and B). On day 5, only one animal from each group exhibited ulceration and acute inflammation, while the others exhibited re epithelization and scanty chronic inflammatory infiltrate. On day 7, none of the specimens exhibited ulcer, but a few exhibited chronic inflammatory infiltrate (Figures 1 C and D). On day 14, all animals were healed. There were no statistically significant differences between groups with regard to ulceration (p = 0.81),inflammation (p = 0.55) or neutrophils (p = 0.53).

Discussion

From the results of the present study, it was observed that oral ulcers treated with PapacarieTM gel exhibited the same inflammatory reaction and healing process as the control group, with ulceration until the third day and remodeling ofthe connective tissue within 14 days. These results corroborate previous findings demonstrating that this gel is biocompatible and may be used in clinical practice.

PapacarieTM has been used on humans as a mechanical chemical method for the removal of carious tissue, as it unites the cleaning and healing properties of papain with the disinfectant characteristics of chloramine. It also helps soften carious dentin tissue, thereby facilitating its removal, as the solution stains only the degraded portion and it does notaffect the healthy tissue surrounding the lesion. Clinical studies have demonstrated favorable results after 1 year of follow-up on 60 teeth in children from 5 to 9 years of age and30 molars in adolescents and adults up to 23 years of age6.

Myagi et al.10 tested the in vitro cytotoxicity of PapacarieTM on human pulp fibroblasts (FP5 cell line). Thegel was applied to round glass coverslips and left in contactwith confluent cultures. Cell viability percentages wereobtained 50 s and 24 h after the cell contact with PapacarieTM. The results showed that direct contact with the gel for 50 s yielded lower cell viability percentages than those of control cells (viable control cells: 100%; viable cells in PapacarieTM group: 80%). After 24 h, this substance achieved the same results as those found in the control group10.

Mastrantonio (2007) analyzed the biocompatibility of papain/chloramine gel on the dorsum of rats and observed a moderate inflammation process in the initial days, comparable to that of the control group, followed by the normal course of healing. These results were reproduced in the present study,in which reepithelization was observed in three days andmoderate inflammatory infiltrate lasted the first five days, followed by the formation of granulation tissue and remodeling of the connective tissue11.

The hypothesis of the present study was that PapacarieTM would accelerate wound closure due to the healing properties of papain and the disinfecting action of chloramine and based on previous favorable clinical results found in vitro, in vivo and on inflamed human pulp tissue6-12. Against expectations,however, the PapacarieTM group behaved like the control group,with no improvement in the oral ulcer healing process. Weused a literature well-established methodology for the studyof oral ulcers, so the size of the ulcer is adequate13. A possible explanation for this outcome is that chloramine in combination with papain alone is not the most adequate agent for the mucosal healing process and perhaps another substance shouldbe added to improve the characteristics of chloramine.

There are a variety of known organic chloramines that have proven useful in organic synthesis. When combined with amino acids, chloramines exhibit interesting biological properties. For example, taurine chloramine has been shown to inhibit the production of superoxide anion (O2-) and nitricoxide (NO) in phagocytes by direct inhibition of the signaling pathways of Ras activation, ERK1/2 phosphorylation andNF-kappaB activation. These effects appear to provide protection from the inadvertent cytotoxicity caused by the overproduction of O2- and NO14-15.

Another hypothesis to explain the lack of statistical differencebetween the groups may be that the PapacarieTM vehicle was not adequate to be used in the oral environment and probably did not stay enough time in the wound. An alternative would beto use an adhesive vehicle as an or a base gel.

In conclusion, the findings of the present study showthat the PapacarieTM gel is biocompatible but, with the presentformulation, it is not useful to improve the healing of oral ulcers.

References
  1. Alternburg A, Zouboulis CC. Current concepts in the treatment of recurrent aphthous stomatitis. Skin Therapy Lett. 2008; 13: 1-4.
  2. Hapa A, Aksoy B, Polat M, Aslan U, Atakan. Does recurrent aphthous stomatitis affect quality of life? A prospective study with 128 patients evaluating different treatment modalities. J Dermatolog Treat. 2010; 22: 380-5.
  3. Payne WG, Salas RE, Ko F, Naidu DK, Donate G, Wright TE et al. Enzymatic debriding agents are safe in wounds with high bacterial bioburdens and stimulate healing. Eplasty. 2008; 8: 17.
  4. da Silva CR, Oliveira MB, Motta ES, de Almeida GS, Varanda LL, de Pádula M et al. Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L.) Using In Vitro Assays. J Biomed Biotechnol. 2010: 197898.
  5. Telgenhoff D, Lam K, Ramsay S, Vasquez V, Villareal K, Slusarewicz P et al. Influence of papain urea copper chlorophyllin on wound matrix remodeling. Wound Repair Regen. 2007; 15: 727-35.
  6. Pereira AS, Silva LR, Motta LJ, Bussadori SK. Remoção química de cárie por meio do gel papacárie. RGO. 2004; 52: 385-8.
  7. Motta LJ, Bussadori SK, Guedes CC, Reda SH, Santos EM. Avaliação in vitro do potencial antimicrobiano de dois sistemas para remoção químicomecânica de dentina cariada: Carisolv e Papacárie. Arq Odontol (UFMG). 2005; 41: 273-368.
  8. Bussadori SK, Fernandes KPS, Martins MD, Guedes CC, Motta LJ, Reda SH et al. Avaliação da biocompatibilidade de um novo material para remoçãoquímica e mecânica da cárie - Papacárie. Pesq Bras Odontoped Clin Integr.2005; 5: 253-59.
  9. Bussadori SK, Castro LC, Galvao AC. Papain gel: a new chemo-mechanical caries removal agent. J Clin Pediatr Dent. 2005; 30: 115-9.
  10. Miagy SPH, Mello I, Bussadori SK, Marques MM. Resposta de fibroblastos humanos em cultura ao gel papacárie. Rev Odontol UNICID. 2006; 18: 245-9.
  11. Mastrantonio SS. Avaliação da biocompatibilidade de materiais para remoção química da lesão de cárie: análise histológica em tecido conjuntivo de camundongos [dissertaton]. Araraquara: Faculdade de Odontologia de Araraquara da UNESP; 2007. 73p.
  12. Martins MD, Fernandes KPS, Motta LJ, Santos EM, Pavesi VCS. Biocompatibility analysis of chemomechanical caries removal material Papacárie on cultured fibroblasts and subcutaneous tissue. J Dent Child (Chic). 2009; 76: 123-9.
  13. Barros, FM; Lotufo, MA; Andrade, PM; França, CM; RC. Possible Association between Th1 Immune Polarization and Epithelial Permeability with Toll-Like Receptors 2 Dysfunction in the Pathogenesis of the Recurrent Aphthous Ulceration. Ulcers. 2010; 1: 1-12.
  14. Kim KS, Park EK, Ju SM, Jung HS, Bang JS, Kim C et al. Taurine chloramin differentially inhibits matrix metalloproteinase 1 and 13 synthesis in interleukin-1beta stimulated fibroblast-like synoviocytes. Arthritis Res Ther. 2007; 9: R80.
  15. Kim C, Cha YN. Production of reactive oxygen and nitrogen species in phagocytes is regulated by taurine chloramin. Adv Exp Med Biol. 2009; 643: 463-72.

Copyright © 2011 - Brazilian Journal of Oral Sciences


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